3-(2-amino-ethyl)-alkylidene)-thiazolidine-2,4-dione and 1-(2-amino-ethyl)-alkylidene-1,3-dihydro-indol-2-one derivatives as selective sphingosine kinase 2 inhibitors

ABSTRACT

3-(2-amino-ethyl)-5-[3-(4-substituted-phenyl)-alkylidene)-thiazolidine-2,4-dione and 1-(2-amino-ethyl)-3-alkylidene-1,3-dihydro-indol-2-one and derivatives thereof are provided for use as selective SphK2 inhibitors and for use in the treatment of human diseases, such as cancer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to 5-alkylidenethiazolidine-2,4-dioneand 3-alkylidene-1,3-dihydro-indol-2-one analogs and their use asselective sphingosine kinase 2 (SphK2) inhibitors in clinicalimplications. In particular, the invention provides5-alkylidenethiazolidine-2,4-dione and3-alkylidene-1,3-dihydro-indol-2-one analogs and derivatives thereof asSphK2 inhibitors and for use in the treatment of cancer.

2. Background of the Invention

Sphingosine-1-phosphate (S1P), a lipid metabolite, has been recognizedand demonstrated as an important signaling mediator for vital cellularand physiological processes, such as cell motility, invasion,proliferation, angiogenesis and apoptosis. S1P is produced fromsphingosine by two kinases, namely, sphingosine kinase 1 (SphK1) andsphingosine kinase 2 (SphK2). Upon production, S1P is secreted andinteracts with a family of G-protein coupled receptors (S1P₁₋₅) on thecell surface to impart a plethora of roles in the regulation of diversephysiological functions such as inflammation, immunity and angiogenesis.Recently, intracellular targets, such as histone deacetylase (HDAC) andTRAF2, have been identified for S1P produced by SphK1 and SphK2,respectively, thus suggesting additional intracellular roles of thissphingolipid metabolite.

S1P and its biosynthetic precursors ceramide and sphingosine are thebest characterized bioactive metabolites of sphingolipids. Ceramide andsphingosine have been associated with growth arrest and apoptosis. Incontrast, S1P has been demonstrated to play important pro-survivalroles. Therefore, the levels of these lipid metabolites need to betightly controlled and a so called sphingolipid rheostat has beenproposed to be crucial in determining cell fate. The regulation of thelevels of these metabolites is complex and a number of enzymes have beendemonstrated to play important roles, among which the SphKs have emergedas a central player in this complex system. SphKs are the key enzymesthat catalyze the production of S1P. To date, two isoenzymes, SphK1 andSphK2 have been identified in human tissues. Although SphK1 and SphK2share a high degree of homology, they have significant differences insize, tissue distribution, and subcellular localization, thus suggestingtheir distinct roles in regulation of different physiological functions.For example, SphK1 is mainly localized in the cytosol while SphK2 ispresent in several intracellular compartments, mainly in the nucleus,endoplasmic reticulum, and mitochondria. Evidence has accumulated thatSphK1 promotes cell growth and survival while the function of SphK2 iscomplex and controversial. Consistent with this notion, numerous studieshave shown that SphK1 is frequently up-regulated and overexpressed intumor tissues compared to normal tissues and SphK1 has been associatedwith many aspects of cancer progression such as proliferation,migration, invasion and angiogenesis. SphK1/S1P has also been implicatedin the pathology of asthma, inflammatory diseases and sepsis. Comparedto SphK1, much less is known about SphK2 and the results arecontrasting. Initially, SphK2 had been demonstrated to be pro-apoptotic.For example, overexpression of SphK2 suppresses growth and promotesapoptosis. However, it has also been demonstrated that downregulation ofSphK2 inhibits the proliferation and migration of tumor cells such asglioblastoma and breast cancer cells. These controversial resultsstrongly suggest developing powerful and selective pharmacological toolsfor SphK2 to better understand the roles of SphK2 in differentpathological conditions. Even though a number of pan-SphK and selectiveSphK1 inhibitors have been developed and reported, the development ofSphK2-selective inhibitors remains limited and SphK2 inhibitors remainscarce, with only a few SphK2 inhibitors having been reported in theliterature (e.g. ABC294620, SG-12, R-FTY720-OMe and trans-12).Therefore, there is an urgent need to develop SphK2-selectiveinhibitors.

SUMMARY OF THE INVENTION

Development of isoform selective SphK inhibitors has attracted extensiveattention as they may serve as valuable pharmacological tools to helpdecipher the physiological and pathological roles of SphKs and aseffective therapeutic agents for human diseases, such as cancer. Eventhough several pan SphK inhibitors and SphK1 selective inhibitors havebeen developed and tested in preclinical experiments, selective SphK2inhibitors remain scarce. During efforts to design and develop novel5-alkylidene-thiazolidine-2,4-dione derivatives as dual-pathwayinhibitors of the Raf/MEK/ERK and PI3K/Akt signaling pathways, a familyof 4-substituted-phenylpropylidene-thiazolidine-2,4-dione analogs werediscovered to be highly selective SphK2 inhibitors. In addition, aseries of 3-alkylidene-1,3-dihydro-indol-2-one analogs has been designedto replace the thiazolidine-2,4-dione heterocycle and they also functionas selective SphK2 inhibitors. Thus, these compounds, depicted ingeneric Formula I and generic Formula II, are selective SphK2 inhibitorsand represent novel therapeutic agents, such as anticancer agents. Inthe Formulas which are depicted herein, the letters N, V, W, X, Y and Zrepresent atoms in the structures of the compounds which may vary asdescribed herein, and do not represent elements such a vanadium,tungsten, etc.

It is an object of this invention to provide a compound of Formula I:

In Formula I, R₁ is selected from the group consisting of: C₃-C₁₄ alkyland C₃-C₁₄ alkoxyl; R₂, R₃, R₄ and R₅ may be the same or different andare independently selected from: H, C₁-C₈ alkyl, C₁-C₈ alkoxyl, C₁-C₈alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; V is S, O,NH, or CH₂; X is C₁-C₄ alkyl; Y is C₁-C₄ alkyl; Z is S or O or NR⁶ inwhich R⁶ is selected from the group consisting of: H, C₁-C₈ alkyl, orisopropyl, or tert-butyl, or a saturated or unsaturated monocyclic ringwith ring size ranging from 3-7 carbons per ring, or unsubstituted orsubstituted phenyl ring which may be substituted with one or moresubstituents selected from the group consisting of: C₁-C₈ alkyl, C₁-C₈alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino, nitro, andcyano; W is NR¹⁶R¹⁷ where R¹⁶ and R¹⁷ may be the same or different andare H; C₁-C₄ alkyl; a saturated heterocycle comprising N bonded directlyto Y; or an unsubstituted or substituted guanidine moiety.

It is also an object of this invention to provide a compound of FormulaII:

wherein, R₁₀ is selected from the group consisting of: C₃-C₁₄ alkyl,C₃-C₁₄ alkoxyl; R₁₁, R₁₂, R₁₃ and R₁₄ may be the same or different andare independently selected from: H, C₁-C₈ alkyl, C₁-C₈ alkoxyl, C₁-C₈alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; R₁₅, R₁₆, R₁₇and R₁₈ may be the same or different and are independently selected fromthe group consisting of: H, C₁-C₈ alkyl, C₁-C₈ alkoxyl, C₁-C₈alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; X is C₁-C₄alkyl; Y is C₁-C₄ alkyl; and W is NR¹⁶R¹⁷ where R¹⁶ and R¹⁷ may be thesame or different and are H; C₁-C₄ alkyl; a saturated heterocyclecomprising N bonded directly to Y; or an unsubstituted or substitutedguanidine moiety.

The invention also provides methods of treating cancer in a patient inneed thereof. The method comprises the step of administering to thepatient a sufficient quantity of a compound of at least one compound ofFormula I:

where, R₁ is selected from the group consisting of: C₃-C₁₄ alkyl andC₃-C₁₄ alkoxyl; R₂, R₃, R₄ and R₅ may be the same or different and areindependently selected from: H, C₁-C₈ alkyl, C₁-C₈ alkoxyl, C₁-C₈alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; V is S, O,NH, or CH₂; X is C₁-C₄ alkyl; Y is C₁-C₄ alkyl; Z is S or O or NR⁶ inwhich R⁶ is selected from the group consisting of: H, C₁-C₈ alkyl, orisopropyl, or tert-butyl, or a saturated or unsaturated monocyclic ringwith ring size ranging from 3-7 carbons per ring, or unsubstituted orsubstituted phenyl ring which may be substituted with one or moresubstituents selected from the group consisting of: C₁-C₈ alkyl, C₁-C₈alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino, nitro, andcyano; and W is NR¹⁶R¹⁷ where R¹⁶ and R¹⁷ may be the same or differentand are H; C₁-C₄ alkyl; a saturated heterocycle comprising N bondeddirectly to Y; or an unsubstituted or substituted guanidine moiety.

In one embodiment of the method, the number of carbon atoms in thealkoxyl substituent of R₁ is 4, 7, or 8. In other embodiments, W is NH₂.The compound may be, for example,3-(2-aminoethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione(Formula III, also referred to herein as “K145” or “compound (30)”) or3-(2-aminoethyl)-5-[3-(4-octoxy-phenyl)-propylidene]-thiazolidine-2,4-dione(Formula IV, also referred to herein as “KLlll16” or “KLlll016” or“compound (31)”).

The invention also provides methods of treating cancer in a patient inneed thereof. The method comprises the step of administering to thepatient a quantity of at least one compound of Formula II sufficient tocure or ameliorate cancer symptoms:

where, R₁₀ is selected from the group consisting of: C₃-C₁₄ alkyl,C₃-C₁₄ alkoxyl; R₁₁, R₁₂, R₁₃ and R₁₄ may be the same or different andare independently selected from: H, C₁-C₈ alkyl, C₁-C₈ alkoxyl, C₁-C₈alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; R₁₅, R₁₆, R₁₇and R₁₈ may be the same or different and are independently selected fromthe group consisting of: H, C₁-C₈ alkyl, C₁-C₈ alkoxyl, C₁-C₈alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; X is C₁-C₄alkyl; Y is C₁-C₄ alkyl; and W is NR¹⁶R¹⁷ where R¹⁶ and R¹⁷ may be thesame or different and are H; C₁-C₄ alkyl; a saturated heterocyclecomprising N bonded directly to Y; or an unsubstituted or substitutedguanidine moiety.

The invention further provides a method of inhibiting the growth orkilling or damaging of human cancer cells. The method comprises the stepof exposing the cell to at least one compound of Formula I and/orFormula II:

In Formula I, R₁ is selected from the group consisting of: C₃-C₁₄ alkyland C₃-C₁₄ alkoxyl; R₂, R₃, R₄ and R₅ may be the same or different andare independently selected from: H, C₁-C₈ alkyl, C₁-C₈ alkoxyl, C₁-C₈alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; V is S, O,NH, or CH₂; X is C₁-C₄ alkyl; Y is C₁-C₄ alkyl; Z is S or O or NR⁶ inwhich R⁶ is selected from the group consisting of: H, C₁-C₈ alkyl, orisopropyl, or tert-butyl, or a saturated or unsaturated monocyclic ringwith ring size ranging from 3-7 carbons per ring, or unsubstituted orsubstituted phenyl ring which may be substituted with one or moresubstituents selected from the group consisting of: C₁-C₈ alkyl, C₁-C₈alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino, nitro, andcyano; and W is NR¹⁶R¹⁷ where R¹⁶ and R¹⁷ may be the same or differentand are H; C₁-C₄ alkyl; a saturated heterocycle comprising N bondeddirectly to Y; or an unsubstituted or substituted guanidine moiety.

In Formula II, R₁₀ is selected from the group consisting of: C₃-C₁₄alkyl, C₃-C₁₄ alkoxyl; R₁₁, R₁₂, R₁₃ and R₁₄ may be the same ordifferent and are independently selected from: H, C₁-C₈ alkyl, C₁-C₈alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino, nitro, andcyano; R₁₅, R₁₆, R₁₇ and R₁₈ may be the same or different and areindependently selected from the group consisting of: H, C₁-C₈ alkyl,C₁-C₈ alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino, nitro, andcyano; X is C₁-C₄ alkyl; Y is C₁-C₄ alkyl; and W is NR¹⁶R¹⁷ where R¹⁶and R¹⁷ may be the same or different and are H; C₁-C₄ alkyl; a saturatedheterocycle comprising N bonded directly to Y; or an unsubstituted orsubstituted guanidine moiety.

In one embodiment of the method, the number of carbon atoms in thealkoxyl substituent of R₁ is 4, 7, or 8. In other embodiments, W is NH₂.The compound may be, for example,3-(2-aminoethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione(Formula III) or3-(2-aminoethyl)-5-[3-(4-octoxy-phenyl)-propylidene]-thiazolidine-2,4-dione(Formula IV).

In yet another embodiment of the method, the cell that is exposed to thecompound is a cancer cell. In some embodiments, the cancer cells areleukemia, lymphoma, sarcoma, neuroblastoma, lung cancer, skin cancer,head squamous cell carcinoma, neck squamous cell carcinoma, prostatecancer, colon cancer, breast cancer, ovarian cancer, cervical cancer,brain cancer, bladder cancer, and/or pancreatic cancer cells.

The invention also provides a method of inhibiting SphK2. The methodcomprises the step of exposing the kinase enzyme to a compound ofFormula I or Formula II:

In Formula I, R₁ is selected from the group consisting of: C₃-C₁₄ alkyland C₃-C₁₄ alkoxyl; R₂, R₃, R₄ and R₅ may be the same or different andare independently selected from: H, C₁-C₈ alkyl, C₁-C₈ alkoxyl, C₁-C₈alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; V is S, O,NH, or CH₂; X is C₁-C₄ alkyl; Y is C₁-C₄ alkyl; Z is S or O or NR⁶ inwhich R⁶ is selected from the group consisting of: H, C₁-C₈ alkyl, orisopropyl, or tert-butyl, or a saturated or unsaturated monocyclic ringwith ring size ranging from 3-7 carbons per ring, or unsubstituted orsubstituted phenyl ring which may be substituted with one or moresubstituents selected from the group consisting of: C₁-C₈ alkyl, C₁-C₈alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino, nitro, andcyano; and W is NR¹⁶R¹⁷ where R¹⁶ and R¹⁷ may be the same or differentand are H; C₁-C₄ alkyl; a saturated heterocycle comprising N bondeddirectly to Y; or an unsubstituted or substituted guanidine moiety.

In Formula II, R₁₀ is selected from the group consisting of: C₃-C₁₄alkyl, C₃-C₁₄ alkoxyl; R₁₁, R₁₂, R₁₃ and R₁₄ may be the same ordifferent and are independently selected from: H, C₁-C₈ alkyl, C₁-C₈alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino, nitro, andcyano; R₁₅, R₁₆, R₁₇ and R₁₈ may be the same or different and areindependently selected from the group consisting of: H, C₁-C₈ alkyl,C₁-C₈ alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino, nitro, andcyano; X is C₁-C₄ alkyl; Y is C₁-C₄ alkyl; and W is NR¹⁶R¹⁷ where R¹⁶and R¹⁷ may be the same or different and are H; C₁-C₄ alkyl; a saturatedheterocycle comprising N bonded directly to Y; or an unsubstituted orsubstituted guanidine moiety.

In one embodiment of the method, the number of carbon atoms in thealkoxyl substituent of R₁ is 4, 7, or 8. In other embodiments, W is NH₂.The compound may be, for example, 3-(2-aminoethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione (Formula III) or3-(2-aminoethyl)-5-[3-(4-octoxy-phenyl)-propylidene]-thiazolidine-2,4-dione(Formula IV).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B. K145 inhibits SphK2 but not SphK1. A) SphK1 and SphK2activities were measured with 5 μM sphingosine in the absence orpresence of the indicated concentrations of K145 or 10 μM DMS. Data areexpressed as percentage SphK activity in the absence of inhibitor; B)Effect of K145 (10 μM) on activity of the indicated enzymes was testedby SelectScreen Kinase Profiling from Invitrogen. CaMKllβ,Ca2+/calmodulin-dependent protein kinase II; CDK2, cyclin-dependentkinase 2; EGFR, Epidermal Growth Factor Receptor; Fyn, Fyn Kinase (p55);ERK2, extracellular signal-regulated kinase 2; ERK1, extracellularsignal-regulated kinase 1; PKA, protein kinase A; PKCα, protein kinaseCα; P13K, phosphatidylinositide 3-kinase. Data are expressed aspercentage of control activity averaged from 2 independent experiments.Data are expressed as mean value ±SEM.

FIG. 2. Compound KLIII16 selectively inhibits SphK2 with a IC₅₀ of 1.9μM

FIG. 3. SphK2 was incubated with indicated compounds at 10 μM, then theactivity of SphK2 was determined. The data shown here is a relativeactivity of SphK2 compared to control (no inhibitor, column labeled Sphin the Figure). 96047 is a known SpK2 inhibitor. A5, B26, K25 and QLA13are known compounds [Li et al. (2009) Bioorg Med Chem Lett 19:6042-6046; Liu et al. (2012) Eur J Med Chem 47: 125-137] and were testedfor purposes of comparison. “A” and “B” refer to different batches of acompound.

FIG. 4. SphK1 was incubated with indicated compounds at 10 μM, then theactivity of SphK1 was determined. The data shown here is a relativeactivity of SphK1 compared to control (no inhibitor, column labeled Sphin the Figure). 96091 is a known Spk1 inhibitor. A5, B26, K25 and QLA13are as above for FIG. 4. “A” and “B” refer to different batches of acompound.

FIG. 5. SphK2 was incubated with indicated compounds at 10 μM, then theactivity of SphK2 was determined. The data shown here is a relativeactivity of SphK2 compared to control (no inhibitor).

FIG. 6. SphK1 was incubated with indicated compounds at 10 μM, then theactivity of SphK2 was determined. The data shown here is a relativeactivity of SphK2 compared to control (no inhibitor).

FIG. 7. Kinetic studies of Formula III (K145) in SphK2 andLineweaver-Burk plot. SphK2 activity was measured with increasingconcentrations of sphingosine and the indicated concentrations of K145.Lineweaver-Burk analysis revealed a Vmax of 10820±210 pmol/min per mg ofprotein, and a K., of 6.4±0.7 μM for SphK2.

FIG. 8. Compound K145 dose-dependently inhibits the growth of humanleukemia U937 cells.

FIG. 9. Compound K145 inhibits the Raf/MEK/ERK and PI3K/Akt signalingpathways.

FIG. 10. Compound S-II-71 and S-II-103 inhibit the Raf/MEK/ERK andPI3K/Akt signaling pathways

FIG. 11. K145 induces apoptosis in U937 cells in dose- andtime-dependent manner.

FIG. 12. S-II-71 and S-II-103 induce apoptosis in U937 cells indose-dependent manner after 24 hrs treatment.

FIG. 13A-D. K145 suppresses the growth of U937 xenograft in nude mice.BALB/c-nu mice (n=7) with palpable U937 xenograft were treated dailywith vehicle, tamibarotene (15 mg/kg), or K145 (15 mg/kg) for 17 days byi.p. injection. A) After treatment, animals were sacrificed and tumorswere removed and weighed and the TGI was calculated; B) Tumor volumeswere measured every other day during the treatment course; C) Animalweights were measured every other day during treatment course. D) Bodyweights of mice throughout treatment. Data are expressed as mean value±SD. *P<0.05 compared to control group.

FIG. 14A-E. K145 suppresses the growth of JC xenograft in BALB/c mice.BALB/c mice (n=8) with palpable JC xenograft were treated daily withvehicle or K145 (20 mg/kg and 35 mg/kg) for 15 days by i.p. injection.A) Tumor volumes were measured every other day; B) After treatment,animals were sacrificed and tumors were removed and weighed; C) The S1Pand K145 levels in the tumor samples from vehicle and treatment (20mg/kg) groups (n=4) were measured by ESI-MS/MS; D) Images of tumorsamples from control and treatment groups (n=7 for each group) after theexperiments; E) Tumor samples (20 mg/kg and control groups) wereanalyzed by Western blot. Data are expressed as mean value ±SEM. *P<0.05compared to control group.

FIG. 15A-D. K145 suppresses the growth of U937 tumors in nude mice byoral administration. BALB/c-nu mice (n=7) with palpable U937 xenograftwere treated daily with vehicle, tamibarotene (20 mg/kg), or K145 (50mg/kg) for 15 days by oral gavage. After treatment, animals weresacrificed and tumors were removed, weighed and images were taken. A)Tumor weight and TGI comparison; B) Images of tumor samples from controland treatment groups (n=7 for each group) after the experiments; C)Tumor volumes were measured every other day; D) Animal weights weremeasured every other day. Data are expressed as mean value ±SEM. *P<0.05compared to control group.

FIG. 16A-E. K145 accumulates and suppresses the S1P level. A and B) U937cells were treated with K145 at the indicated concentrations for 3 h andthe levels of K145 and S1P were measured by ESI-MS/MS. C) HEK293 cellswere treated with K145 (10 μM) for 2 h. Lipids were extracted anddifferent chain length species of ceramide were determined byLC-ESI-MS/MS. Numbers indicate chain length followed by the number ofdouble bonds in the fatty acid. Data are averages of triplicatedeterminations and are expressed as pmol lipid/10⁶ cells. D) U937 cellswere treated with or without K145 (10 μM) for 3 h and levels of C1Pspecies were determined by ESI-MS/MS. E) U937 cells were treated withFTY720 (1 μM) in the absence or presence of indicated K145 for 3 h, thenFTY720-P was measured by ESI-MS/MS. *P<0.05 compared to control.

DETAILED DESCRIPTION

The invention provides compounds of the following Formula I and FormulaII:

In Formula I:

R₁ is selected from the group consisting of: C₃-C₁₄ alkyl and C₃-C₁₄alkoxyl;

R₂, R₃, R₄ and R₅ may be the same or different and are independentlyselected from: H, C₁-C₈ alkyl, C₁-C₈ alkoxyl, C₁-C₈ alkylcarbonyl,halogen, hydroxyl, amino, nitro, and cyano;

V is S, O, NH, or CH₂;

X is C₁-C₄ alkyl;

Y is C₁-C₄ alkyl;

Z is S or O or NR⁶ in which R⁶ is selected from the group consisting of:H, C₁-C₈ alkyl, or isopropyl, or tert-butyl, a saturated or unsaturatedmonocyclic ring with ring size ranging from 3-7 carbons per ring, anunsubstituted or substituted phenyl ring which may be substituted withone or more substituents selected from the group consisting of: C₁-C₈alkyl, C₁-C₈ alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino,nitro, and cyano; and

W is NR₇R₈ where R₇ and R₈ may be the same or different and areindependently selected from H; C₁-C₄ alkyl; a saturated heterocyclecomprising N bonded directly to Y; and an unsubstituted or substitutedguanidine moiety.

In Formula II:

R₁₀ is selected from the group consisting of: C₃-C₁₄ alkyl, C₃-C₁₄alkoxyl;

R₁₁, R₁₂, R₁₃ and R₁₄ may be the same or different and are independentlyselected from: H, C₁-C₈ alkyl, C₁-C₈ alkoxyl, C₁-C₈ alkylcarbonyl,halogen, hydroxyl, amino, nitro, and cyano;

R₁₅, R₁₆, R₁₇ and R₁₈ may be the same or different and are independentlyselected from the group consisting of: H, C₁-C₈ alkyl, C₁-C₈ alkoxyl,C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano;

X is C₁-C₄ alkyl;

Y is C₁-C₄ alkyl;

and

W is NR¹⁶R¹⁷ where R¹⁶ and R¹⁷ may be the same or different and are H;C₁-C₄ alkyl; a saturated heterocycle comprising N bonded directly to Y;or an unsubstituted or substituted guanidine moiety.

By “saturated heterocycle” we mean a saturated monocyclic carbon ringcontaining at least one heteroatom atom N as part of the ring. Themonocyclic ring is fully saturated (i.e. it does not contain anycarbon-carbon double or triple bonds). In addition to N bonded directlyto Y, one or more additional positions in the ring(s) may be substitutedby other heteroatoms, examples of which include but are not limited to:N, O, S, etc. Exemplary saturated heterocycles that may be used in thepractice of the invention include but are not limited to morpholine,piperidine, piperazine, pyrrolidine, etc.

By “saturated or unsaturated monocyclic ring” we mean a fully saturatedmonocyclic carbon ring (i.e. it does not contain any carbon-carbondouble or triple bonds) without or with at least one heteroatom,examples of which include but are not limited to: one or more N, O, S,etc; as part of the ring. Unsaturated monocyclic ring means a monocycliccarbon ring containing one or more carbon-carbon or carbon-heteroatomdouble or triple bonds) without or with at least one heteroatom,examples of which include but are not limited to: one ore more N, O, S,etc; as part of the ring.

C₁-C₄ alkyl includes include substituents with 1, 2, 3, or 4 carbonatoms and may be unbranched or branched isomeric forms thereof, e.g.methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl; C₁-C₈ alkyl, C₁-C₈ alkoxyl, and C₁-C₈ alkylcarbonyl includesubstitutents with 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms in the alkylportion of the molecule (which may be unbranched or branched isomericforms thereof), e.g. they comprise methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl,hexyl, 2-methylpentane, 3-methylpentane, 2,3-dimethylbutane,2,2-dimethylbutane and various 8-carbon octyl component and branchedisomers thereof, etc., as understood in the art.

In one embodiment of the invention, the compound of Formula I is thecompound3-(2-aminoethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dioneshown in Formula III. In another embodiment of the invention, thecompound of Formula I is3-(2-aminoethyl)-5-[3-(4-octoxy-phenyl)-propylidene]-thiazolidine-2,4-dioneshown in Formula IV. The compounds of Formulas III and IV inhibit SphK2selectively, i.e. they do not inhibit SphK1, or they inhibit SphK2 to agreater extent, e.g. inhibition of SphK2 is at least about 2 fold, andusually about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100-fold or moregreater than is inhibition of SphK1.

The synthesis and structural characterization of the compoundsrepresented by Formula III and Formula IV are described in Examples 3,10 and 15.

Other compounds that inhibited SphK2 include: S-11-73, compound (61);KL11016, compound (31); and KL111023, compound (33); (see FIGS. 3 and 4for activity and Example 8 for structures of (61), (31), (33); andKL11139, compound (34)]; KL1147, compound (35); KL157, compound (32);S-11-103, compound (66); and S-11-104, compound (70); (see FIGS. 5 and 6for activity and Example 8 for structures of (34), (35), (32), (66),(70)). These compounds inhibited SphK2 selectively, compared to Sphk1,i.e. they did not inhibit SphK1 or inhibited SphK1 to a lesser extent asdescribed above.

Compound K11167 [compound (42) herein] inhibits both Sphk1 and Sphk2(see FIGS. 5 and 6 for activity and Example 8 for the structure ofcompound (42)), and may be used for the non-selective inhibition of oneor both of the enzymes.

The invention also provides compositions for the treatment of diseasesor conditions associated with the over-activation or over-expression ofSphK2. In particular, the invention provides compositions for thetreatment of various cancers. The compositions comprise at least onecompound of Formula I and/or at least one compound of Formula II and apharmaceutically acceptable (i.e. a physiologically compatible) carrier,e.g. saline, pH in the range of about 6.5 to about 7.5, and usuallyabout 7.2). Depending on the route of administration, the compositionscan take the form of liquids suitable for injection or intravenousadministration, aerosols, cachets, capsules, creams, elixirs, emulsions,foams, gels, granules, inhalants, liposomes, lotions, magmas,microemulsion, microparticles, ointments, peroral solids, powders,sprays, syrups, suppositories, suspensions, tablets and tinctures. Theamount of the compound of Formula 1 and/or Formula II present in thecomposition can vary, but us usually in the range of from about 1 to99%.

The compositions may include one or more pharmaceutically compatibleadditives or excipients. Commonly used pharmaceutical additives andexcipients which can be used as appropriate to formulate the compositionfor its intended route of administration include but are not limited to:

acidifying agents (examples include but are not limited to acetic acid,citric acid, fumaric acid, hydrochloric acid, nitric acid);alkalinizing agents (examples include but are not limited to ammoniasolution, ammonium carbonate, diethanolamine, monoethanolamine,potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide,triethanolamine, trolamine);adsorbents (examples include but are not limited to powdered celluloseand activated charcoal);aerosol propellants (examples include but are not limited to carbondioxide, CCl₂F₂, F₂ClC—CClF₂ and CClF₃);air displacement agents (examples include but are not limited tonitrogen and argon);antifungal preservatives (examples include but are not limited tobenzoic acid, butylparaben, ethylparaben, methylparaben, propylparaben,sodium benzoate, propionic acids or its salts);antimicrobial preservatives (examples include but are not limited tobenzalkonium chloride, benzethonium chloride, benzyl alcohol,cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol,phenylmercuric nitrate and thimerosal);antioxidants (examples include but are not limited to ascorbic acid,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorus acid, monothioglycerol, propyl gallate, sodium ascorbate,sodium bisulfate, sodium formaldehyde sulfoxylate, sodium metabisulfite,tocopherol, and vitamin E);binding materials (examples include but are not limited to blockpolymers, natural and synthetic rubber, polyacrylates, polyurethanes,silicones and styrene-butadiene copolymers);buffering agents (examples include but are not limited to potassiummetaphosphate, potassium phosphate monobasic, sodium acetate, sodiumcitrate anhydrous and sodium citrate dihydrate);carrying agents (examples include but are not limited to acacia syrup,aromatic syrup, aromatic elixir, cherry syrup, cocoa syrup, orangesyrup, syrup, corn oil, mineral oil, peanut oil, sesame oil,bacteriostatic sodium chloride injection and bacteriostatic water forinjection);chelating agents (examples include but are not limited to edetatedisodium and edetic acid); colorants (examples include but are notlimited to FD&C Red No. 3, FD&C Red No. 20, FD&C Yellow No. 6, FD&C BlueNo. 2, D&C Green No. 5, D&C Orange No. 5, D&C Red No. 8, caramel, ferricoxide red, natural colorants such as bixin, norbixin, and carmine);clarifying agents (examples include but are not limited to bentonite);emulsifying agents (examples include but are not limited to acacia,cetomacrogol, cetyl alcohol, glyceryl monostearate, lecithin, sorbitanmonooleate, and polyethylene 50 stearate);encapsulating agents (examples include but are not limited to gelatinand cellulose acetate phthalate);fillers (examples include but are not limited to sugars, lactose,sucrose, sorbitol, cellulose preparations, calcium phosphates, naturalor synthetic gums, solid starch, and starch pastes); flavorants(examples include but are not limited to anise oil, cinnamon oil, cocoa,menthol, orange oil, peppermint oil and vanillin);humectants (examples include but are not limited to glycerin, propyleneglycol and sorbitol);levigating agents (examples include but are not limited to mineral oiland glycerin);oils (examples include but are not limited to arachis oil, mineral oil,olive oil, peanut oil, sesame oil and vegetable oil);ointment bases (examples include but are not limited to lanolin,hydrophilic ointment, polyethylene glycol ointment, petrolatum,hydrophilic petrolatum, white ointment, yellow ointment, and rose waterointment);penetration enhancers (transdermal delivery) (examples include but arenot limited to monohydroxy or polyhydroxy alcohols, saturated orunsaturated fatty alcohols, saturated or unsaturated fatty esters,saturated or unsaturated dicarboxylic acids, essential oils,phosphatidyl derivatives, cephalin, terpenes, amides, ethers, ketonesand ureas);plasticizers (examples include but are not limited to diethyl phthalateand glycerin);solvents (examples include but are not limited to alcohol, corn oil,cottonseed oil, glycerin, isopropyl alcohol, mineral oil, oleic acid,peanut oil, purified water, water for injection, sterile water forinjection and sterile water for irrigation);stiffening agents (examples include but are not limited to cetylalcohol, cetyl esters wax, microcrystalline wax, paraffin, stearylalcohol, white wax and yellow wax);suppository bases (examples include but are not limited to cocoa butterand polyethylene glycols (PEGS) (and mixtures containing one or both ofcocoa butter and PEGs));surfactants (examples include but are not limited to benzalkoniumchloride, nonoxynol 10, oxtoxynol 9, polysorbate 80, sodium laurylsulfate and sorbitan monopalmitate);suspending agents (examples include but are not limited to agar,bentonite, carbomers, carboxymethylcellulose sodium, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,kaolin, methylcellulose, tragacanth and veegum);sweetening agents (examples include but are not limited to aspartame,dextrose, fructose, glycerin, mannitol, propylene glycol, saccharinsodium, sorbitol and sucrose);tablet anti-adherents (examples include but are not limited to magnesiumstearate and talc); tablet binders (examples include but are not limitedto acacia, alginic acid, carboxymethylcellulose sodium, compressiblesugar, ethylcellulose, gelatin, liquid glucose, methylcellulose,povidone and pregelatinized starch);tablet and capsule diluents (examples include but are not limited todibasic calcium phosphate, kaolin, lactose, mannitol, microcrystallinecellulose, powdered cellulose, precipitated calcium carbonate, sodiumcarbonate, sodium phosphate, sorbitol and starch);tablet coating agents (examples include but are not limited to liquidglucose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, methylcellulose, ethylcellulose, cellulose acetatephthalate and shellac);tablet direct compression excipients (examples include but are notlimited to dibasic calcium phosphate);tablet disintegrants (examples include but are not limited to alginicacid, carboxymethylcellulose calcium, microcrystalline cellulose,polacrillin potassium, sodium alginate, sodium starch glycollate andstarch);tablet glidants (examples include but are not limited to colloidalsilica, corn starch and talc);tablet lubricants (examples include but are not limited to calciumstearate, magnesium stearate, mineral oil, stearic acid and zincstearate);tablet/capsule opaquants (examples include but are not limited totitanium dioxide);tablet polishing agents (examples include but are not limited to carnubawax and white wax);thickening agents (examples include but are not limited to beewax, cetylalcohol and paraffin);tonicity agents (examples include but are not limited to dextrose andsodium chloride);viscosity increasing agents (examples include but are not limited toalginic acid, bentonite, carbomers, carboxymethylcellulose sodium,methylcellulose, povidone, sodium alginate and tragacanth); andwetting agents (examples include but are not limited toheptadecaethylene oxycetanol, lecithins, polyethylene sorbitolmonooleate, polyoxyethylene sorbitol monooleate, polyoxyethylenestearate).

Additional additives and excipients suitable for pharmaceutical use suchas those described in Remington's The Science and Practice of Pharmacy,21^(st) Edition (2005), Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 11^(th) Edition (2005) and Ansel's Pharmaceutical DosageForms and Drug Delivery Systems (8^(th) Edition), edited by Allen etal., Lippincott Williams & Wilkins, (2005) are also considered to bewithin the scope of the invention. The complete contents of thesereferences are herein incorporated by reference in entirety.

In one embodiment of the compositions of the invention, one or more(i.e. at least one) additional anti-cancer agent can be added to thecomposition. Representative anti-cancer agents include, but are notlimited to, Erbitux, methotrexate, taxol, mercaptopurine, thioguanine,hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas,cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine,etoposides, campathecins, bleomycin, doxorubicin, idarubicin,daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase,vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel,γ-radiation, alkylating agents including nitrogen mustard such ascyclophosphamide, ifosfamide, trofosfamide, chlorambucil, nitrosoureassuch as carmustine (BCNU), and lomustine (CCNU), alkylsulphonates suchas busulfan, and treosulfan, triazenes such as dacarbazine, platinumcontaining compounds such as cisplatin and carboplatin, plant alkaloidsincluding vinca alkaloids, vincristine, vinblastine, vindesine, andvinorelbine, taxoids including paclitaxel, and docetaxol, DNAtopoisomerase inhibitors including epipodophyllins such as etoposide,teniposide, topotecan, 9-aminocamptothecin, campto irinotecan, andcrisnatol, mitomycins such as mitomycin C, anti-metabolites, includinganti-folates such as DHFR inhibitors, methotrexate and trimetrexate, IMPdehydrogenase inhibitors including mycophenolic acid, tiazofurin,ribavirin, EICAR, ribonucleotide reductase inhibitors such ashydroxyurea, deferoxamine, pyrimidine analogs including uracil analogs5-fluorouracil, floxuridine, doxifluridine, and ratitrexed, cytosineanalogs such as cytarabine (ara C), cytosine arabinoside, andfludarabine, purine analogs such as mercaptopurine, thioguanine,hormonal therapies including receptor antagonists, the anti-estrogenstamoxifen, raloxifene and megestrol, LHRH agonists such as goscrclin,and leuprolide acetate, anti-androgens such as flutamide, andbicalutamide, retinoids/deltoids, Vitamin D3 analogs including EB 1089,CB 1093, and KH 1060, photodyamic therapies including vertoporfin(BPD-MA), phthalocyanine, photosensitizer Pc4, Demethoxy-hypocrellin A,(2BA-2-DMHA), cytokines including Interferon, α-Interferon,γ-interferon, tumor necrosis factor, as well as other compounds havinganti-tumor activity including isoprenylation inhibitors such aslovastatin, dopaminergic neurotoxins such as 1-methyl-4-phenylpyridiniumion, cell cycle inhibitors such as staurosporine, alsterpaullone,butyrolactone I, Cdk2 inhibitor, Cdk2/Cyclin Inhibitory Peptide I,Cdk2/Cyclin Inhibitory Peptide II, Compound 52[2-(2-hydroxyethylamino)-6-(3-chloroanilino)-9-isopropylpurine],Indirubin-3′-monoxime, Kenpaullone, Olomoucine, Iso-olomoucine,N⁹-isopropyl-olomoucine, Purvalanol A, Roscovitine, (5)-isomerRoscovitine and WHI-P180[4-(3′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, actinomycins suchas actinomycin D and dactinomycin, bleomycins such as bleomycin A2,bleomycin B2, and peplomycin, anthracyclines such as daunorubicin,doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin,zorubicin, and mitoxantrone, MDR inhibitors including verapamil, andCa²⁺ ATPase inhibitors such as thapsigargin.

In addition, the compounds or compositions of the invention may beadministered in conjunction with other health-related and/or cancertreating substances or protocols, including but not limited to: dietarymodifications (e.g. vitamin or antioxidant therapy); pain medication orprocedures to lessen pain; radiation; various forms of chemotherapy(e.g. administration of platinum drugs, etc.; surgery; cryotherapy; andmedications to lessen nausea, etc.).

The invention also provides methods of treating cancer in a patient inneed thereof. The methods comprise a step of administering, to thepatient, an effective amount of one or more compounds of Formulas Iand/or II, e.g. as a composition comprising the compound(s). Methods ofadministration include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous (IV), intratumoral,subcutaneous, intranasal, epidural, oral, sublingual, intranasal,intracerebral, intravaginal, transdermal, rectally, by inhalation, ortopically, particularly to the ears, nose, eyes, or skin. Frequently,administration will be IV, although the mode of administration is leftto the discretion of the skilled practitioner (e.g. a physician). Inmost instances, administration will result in the release of a compoundof the invention into the bloodstream. However, this need not always bethe case, e.g. with topical or intratumoral administration. Further,modes of administration may be combined, e.g. intravenous andintratumoral administration may both be carried out in a patient.

The amount of the compound(s) of Formulas I and/or II that isadministered in one administration is generally in the range of fromabout 0.1 to about 10 mg/kg of body weight of the patient, and isusually in the range of from about 0.1 to about 10 mg/kg, with a goal ofachieving levels of from about 1 to about 5 μM in the blood stream.Those of skill in the art will recognize that administration may becarried out according to any of several protocols, and will generally bedetermined by a skilled practitioner such as a physician. For example,administration may be once per day, several times per day, or lessfrequent (e.g. weekly, biweekly, etc.). The amount of the compound thatis administered and the frequency of administration may depend onseveral factors, e.g. the characteristics of the patient (weight, age,gender, overall state of health, etc.); the type and stage of the cancerbeing treated; the response of the patient to the treatment; etc.

By “an effective amount” we mean an amount that is sufficient toameliorate, lessen or eliminate symptoms of the disease that is beingtreated. While in some cases, the patient may be completely “cured”(disease symptoms disappear entirely), this need not always be the case.Those of skill in the art will recognize that substantial benefits mayaccrue if disease symptoms are only partially mitigated, or if theprogress of the disease is slowed. For example, when treating cancer,substantial benefits re quality of life and longevity are obtained byslowing or arresting the growth of a tumor and/or preventing metastasis,shrinking (decreasing) the size of a tumor, etc. even if the tumoritself is not entirely destroyed by exposure to the compounds describedherein. In some cases, the cancer cells which are exposed to thecompounds of the invention are killed; in other embodiments, the cancercells are damaged, e.g. prevented from growing or rendered incapable ofcell division, etc.

Types of cancer that can be treated using the compounds and methodsdescribed herein include but are not limited to: leukemia, lymphoma,sarcoma, neuroblastoma, lung cancer, skin cancer, squamous cellcarcinoma of the head and neck, prostate cancer, colon cancer, breastcancer, ovarian cancer, cervical cancer, brain cancer, bladder cancer,pancreatic cancer. The cancer may be at any stage of development, andpre-cancerous cells may also be treated.

The patient or subject that is treated in this manner is usually amammal, although this is not always the case. Frequently, the mammal isa human, although the methods may also be applied to the treatment ofother animals, e.g. in veterinary practice.

The invention also provides methods of inhibiting SphK2 and downstreamsignaling pathways in a cell. In some embodiments, inhibition of SphK2is selective, e.g. the compound inhibits SphK2 but not other enzymes,for example, SphK1. In this embodiment, compound K11167 is excludedsince it inhibits both SphK2 and SphK1. The methods involve exposing thecells to one or more compounds of the invention, the one or morecompounds being present in an amount that is sufficient to inhibit theenzyme, SphK2, usually by at least 50%, in some cases by 60%, 70%, 80%,90%, 95% or more, or even completely (i.e. 100% inhibition), compared toan untreated control. Those of skill in the art are familiar withmethods to measure levels of inhibition of SphK2, e.g. by detecting theamount of a metabolite of SphK2 substrate or compound that participatesin the downstream signaling pathway or that is made by or in thepathway, e.g. by measuring an amount or degree of mRNA or proteinexpression, or the amount of protein modification (e.g. phosphorylationor de-phosphorylation), etc. In some cases, the cells in which thesepathways are inhibited are cancer cells.

The invention also provides methods of inhibiting SphK2. In someembodiments, inhibition of SphK2 is selective, e.g. the compoundinhibits SphK2 but not other enzymes, for example, SphK1. In thisembodiment, compound Klll67 is excluded since it inhibits both SphK2 andSphK1 (see FIGS. 5 and 6). The methods of the invention involve bringingthe enzyme into contact with one or more compounds of the invention,e.g. by contacting, exposing or otherwise providing access of thecompound(s) to the enzyme(s). The kinase may be an isolated purified orpartially purified enzyme, or may be within a cell (e.g. in a cellcultured in vitro), or within and organism (in vivo). Generally, theactivity of the kinase is inhibited by at least about 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% or even more, e.g.about 100%, compared to a control enzyme which is not exposed to acompound of the invention. Those of skill in the art are familiar withmethodology to measure the activity of enzymes, and of kinases inparticular. For example, the ability of a kinase to carry out its usualenzymatic activity may be measured, e.g. by detecting a product of thatactivity.

The invention also provides methods of inhibiting SphK1 and SphK2 usingthe compound Klll67 (compound 42; see the Scheme in the Examplesentitled “Compounds 28-36, 39-40 and 42 of Schemes 2 and 3”). Compound42 may be used for any of the purposes described herein, e.g. forinhibiting SphK2, for treating diseases associated with SphK2, forkilling or damaging cancer cells, for treating cancer, etc. However, itsuse is not selective for SphK2. Thus, compound 42 may also be used toinhibit SphK1, to treat diseases or conditions associated with SphK1,etc. In further embodiments, compound 42 may be used to inhibit bothSphK1 and SphK2, e.g. in vitro or in cells.

The invention also provides methods of inhibiting growth or killing ordamaging cells exhibiting positive SphK2 activity. By “positive” SphK2activity we mean overactivation or overexpression of the kinase. In someembodiments, the cells are cancer cells. The methods involve exposingthe cells to one or more compounds of the invention, the one or morecompounds being present in an amount that is sufficient to cause thedeath of the cells, or to cause damage to the cells, e.g. to slow thecells' metabolism, prevent replication, prevent movement, induceapoptosis of the cells, etc. The cells that are killed or damaged may bein vitro or in vivo, i.e. this method may be carried out for clinicalpurposes (e.g. for the treatment of disease) or in the laboratory (e.g.the compounds of the invention may be used as laboratory reagents.) Whena population of cancer cells is exposed to the compounds of theinvention, generally about 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or more, e.g. even 100%, or the cells in the population aredamaged or killed, compared to a suitable control population that is notexposed to the compounds.

Other embodiments of the invention include the treatment of diseases orconditions associated with positive SphK2 activity. These methodscomprise the step of administering an effective amount of the compoundof Formula I and/or Formula II or a composition thereof to a patient inneed thereof to inhibit the SphK2 activity. Examples of such disease orconditions include but are not limited to cancer, arthrosclerosis,arthritis, diabetes, obesity, osteoporosis, inflammatory diseases andAlzheimer's disease. An “effective amount” or a “therapeutic amount”refers to an amount that either cures (i.e. symptoms of diseasedisappear completely or become undetectable), or ameliorates diseasesymptoms, e.g. by lessening symptoms such as pain, slowing of the growthof a tumor, lengthening the life of a patient, improving the quality oflife of a patient, etc.

The invention is further described by the following non-limitingexamples which further illustrate the invention, and are not intended,nor should they be interpreted to, limit the scope of the invention inany way.

EXAMPLES

The synthesis of the exemplary compounds is described in Schemes 1-5.

Example 1 Preparation of Compound 26

To a stirred suspension of bromoethylamine hydrobromide 25 (20.5 g, 100mmol), (Boc)₂O (21.8 g, 100 mmol) in dichloromethane (200 mL) was addedtriethylamine (13.9 mL, 100 mmol) dropwise at 0° C., after added themixture was stirred at room temperature (rt). overnight, water wasadded, the separated CH₂Cl₂ layer was washed with brine, and dried byNa₂SO₄, remove the solvent to give a colorless oil (20 g).

Example 2 Preparation of Compound 27

A mixture of 2,4-thiazolidinedione (7.9 g, 68 mmol), compound 26 (17.9g, 80 mmol), K₂CO₃ (11.1 g, 92 mmol), TBAI (2.5 g, 6.8 mmol) in acetone(100 mL) was stirred at 40° C. for 10 h, suction filter, the filtratewas concentrated under vacuum, the residue was purified by flash columnchromatography (Hexane/EA=4/1 to 2/1) to give a white solid (12.3 g).

Example 3 Preparation of Compounds 28-36

A solution of compound 27 (12 mmol), aldehyde (12 mmol) and piperidine(3.6 mmol) in MeOH (60 mL) was stirred at rt. overnight, remove solventunder vacuum, the residue was purified by flash column chromatography(Hexane/EA=8/1) to give a white solid, which was subject to Bocdeprotection conditions in ethyl acetate (30 mL) by 4 M HCl in dioxane(15 mL), the solution to give 28-36 as white solid.

Example 4 Preparation of 4-alkoxyphenylpropanal Compounds 14-19

To a stirred solution of compounds 2-7 (891 mg, 5 mmol) and meldrum'sacid (720 mg, 5 mmol) in EtOH (5 mL) was added piperidine (two drops),the resulting solution was stirred at rt. overnight, suction filter givea light yellow solid, (731 mg). To the solid and acetic acid (3 mL) inDCM (20 mL) at 0° C. was added sodium borohydride (314 mg, 8.4 mmol)portion wise. The resulting solution was stirred at rt. for 1 h. thesolution was dissolved in DCM and washed with brine and water. Theorganic layer was dried over Na₂SO₄ and concentrated under vacuum, theresidue was purified by flash column chromatography (Hexane/acetone=5/2)to give a light yellow solid (426 mg). To a solution of above compounds(368 mg, 1.2 mmol) in tetrahydrofuran (7 mL) was added triethylamine(0.334 mL, 2.4 mmol) followed by phenylsilane (0.444 mL, 3.6 mmol). Theresulting solution was stirred for 2 hours at room temperature. Waterwas added to the solution and stirred for 15 minutes. The reactionmixture was dissolved in ether and washed with water, then with brine.The organic layer was dried over Na₂SO₄ and concentrated, the residuewas purified by flash column chromatography (Hexane/acetone=10/1) togive a light colorless oil.

Example 5 Preparation of 4-alkoxybenzaldehyde Compounds 2-7

A mixture of 4-hydroxybenzaldehyde (10 mmol), bromoalkane (13 mmol),K₂CO₃ (13 mmol), DMF (20 mL) was refluxed for 18 h, cooled to rt., waterwas added, extracted by hexane, concentrated under vacuum, the residuewas purified by flash column chromatography to give a light yellow oil.

Example 6

Preparation of Compounds 24, 46, 48 and 50 by Swern Oxidation

DMSO (14 mmol) was added dropwise to a stirred solution of oxalylchloride (5 mmol) in DCM (20 mL) at −78° C., after added the mixture wasstirred at −78° C. for 20 min, then alkanol (4 mmol) was added dropwiseand stirred at −78° C. for 1 h, Et₃N (1 mL) was added and thetemperature was slowly elevated to it, then water was added, theseparated DCM layer was washed with brine, concentrated under vacuum,the residue was purified by flash column chromatography to give acolorless oil.

Example 7 Preparation of Compound 39 by Reduction

A mixture of compound 31 (KLIII016) (100 mg) and 10% Pd/C (50 mg) inmethanol (15 mL) was hydrogenated at rt. overnight, suction filter toremove the catalyst, the filtrate was concentrated under vacuum, thenethyl acetate (1 mL) was added, suction filter, and washed with ethylacetate to give a white solid.

Example 8 Preparation of Guanidine Analog 42

To a suspension of 31 (KLIII016) (0.2 mmol) in DCM (2 mL) was addedtriethylamime (0.2 mmol) followed byN,N′-Di-Boc-1H-pyrazole-1-carboxamidine (0.2 mmol), after added themixture was stirred at rt. overnight, water was added and extracted byDCM, concentrated under vacuum, the residue was purified by flashchromatography (Hexane/Acetone=10/1) give 41 as a colorless oil (88 mg).Compound 41 (80 mg) was dissolved in 2 mL of DCM, 2 mL of HCl (4M indioxane) was added, the mixture was stirred at rt. for 20 h, filtered togive 28 mg of compound 42 as white solid.

Compounds 28-36 39-40 and 42 of Schemes 2 and 3

Compounds 59-70 of Scheme 5

Example 9 KII167 (42)

¹H NMR (400 MHz, DMSO-d₆): 7.62 (m, 1H), 7.34 (brs, 3H), 7.15-7.13 (d,J=8.5 Hz, 2H), 7.01 (t, J=7.4 Hz, 1H), 6.86-6.83 (d, J=8.5 Hz, 2H), 3.91(t, J=6.5 Hz, 2H), 3.67 (t, J=4.9 Hz, 2H), 3.39-3.35 (q, J=5.9 Hz, 2H),2.76 (t, J=7.3 Hz, 2H), 2.53-2.45 (m, 2H), 1.72-1.65 (m, 2H), 1.41-1.39(m, 2H), 1.37-1.28 (m, 6H), 0.87 (t, J=6.6 Hz, 3H); ¹³C NMR (100 MHz,DMSO-d₆): 167.3, 164.3, 157.1, 156.9, 137.5, 132.0, 129.3, 125.1, 114.4,67.3, 40.5, 38.2, 33.0, 32.1, 31.2, 28.7, 28.4, 25.5, 22.0, 13.9.

Example 10 KLIII16 (31)

¹H NMR (400 MHz, DMSO-d₆): 8.13 (s, 3H), 7.15-7.13 (d, J=8.6 Hz, 2H),7.01 (t, J=7.4 Hz, 1H), 6.85-6.83 (d, J=8.6 Hz, 2H), 3.91 (t, J=6.5 Hz,2H), 3.83 (t, J=6.0 Hz, 2H), 3.00 (m, 2H), 2.76 (t, J=7.4 Hz, 2H),2.53-2.45 (m, 2H), 1.70-1.66 (m, 2H), 1.41-1.37 (m, 2H), 1.35-1.26 (m,6H), 0.87 (t, J=6.8 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆): 167.6, 164.5,157.1, 137.4, 132.0, 129.2, 125.2, 114.4, 67.3, 36.5, 33.0, 32.1, 31.2,28.7, 28.4, 25.5, 22.0, 13.9.

Example 11 KLII157 (32)

¹H NMR (400 MHz, DMSO-d₆): 8.16 (s, 3H), 7.15-7.13 (d, J=8.6 Hz, 2H),7.01 (t, J=7.4 Hz, 1H), 6.85-6.83 (d, J=8.6 Hz, 2H), 3.91 (t, J=6.5 Hz,2H), 3.83 (t, J=6.0 Hz, 2H), 3.00 (m, 2H), 2.76 (t, J=7.4 Hz, 2H),2.53-2.45 (m, 2H), 1.70-1.64 (m, 2H), 1.40-1.35 (m, 2H), 1.32-1.25 (m,10H), 0.86 (t, J=6.8 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆): 167.6, 164.5,157.1, 137.4, 132.0, 129.2, 125.2, 114.4, 67.3, 36.5, 33.0, 32.1, 31.2,28.9, 28.7, 28.6, 28.5, 25.5, 22.0, 13.9.

Example 12 KLII123 (33)

¹H NMR (400 MHz, DMSO-d₆): 8.11 (s, 3H), 7.15-7.13 (d, J=8.5 Hz, 2H),7.01 (t, J=7.4 Hz, 1H), 6.85-6.83 (d, J=8.5 Hz, 2H), 3.91 (t, J=6.5 Hz,2H), 3.83 (t, J=6.0 Hz, 2H), 3.00 (m, 2H), 2.76 (t, J=7.4 Hz, 2H),2.53-2.48 (m, 2H), 1.69-1.66 (m, 2H), 1.40-1.37 (m, 2H), 1.27-1.25 (m,14H), 0.85 (t, J=6.8 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆): 167.6, 164.5,157.1, 137.4, 132.0, 129.2, 125.2, 114.4, 67.3, 36.6, 33.0, 32.1, 31.2,28.9, 28.7, 28.68, 28.65, 25.5, 22.0, 13.9.

Example 13 KLII147 (35)

¹H NMR (400 MHz, DMSO-d₆): 8.12 (s, 3H), 7.03 (t, J=7.4 Hz, 1H), 3.85(t, J=6.0 Hz, 2H), 3.02 (m, 2H), 2.24-2.19 (q, J=7.4 Hz, 2H), 1.51-1.48(m, 2H), 1.31-1.24 (m, 20H), 0.86 (t, J=6.8 Hz, 3H); ¹³C NMR (100 MHz,DMSO-d₆): 167.6, 164.6, 138.3, 124.9, 36.6, 31.3, 31.1, 29.0, 28.9,28.8, 28.7, 28.6, 27.2, 22.0, 13.9.

Example 14 KLII139 (34)

¹H NMR (400 MHz, DMSO-d₆): 8.10 (s, 3H), 7.15-7.13 (d, J=8.2 Hz, 2H),7.12-7.10 (d, J=8.2 Hz, 2H), 7.02 (t, J=7.4 Hz, 1H), 3.83 (t, J=6.0 Hz,2H), 3.00 (m, 2H), 2.79 (t, J=7.4 Hz, 2H), 2.55-2.49 (m, 4H), 1.55-1.51(m, 2H), 1.40-1.37 (m, 2H), 1.26-1.23 (m, 10H), 0.85 (t, J=6.8 Hz, 3H);¹³C NMR (100 MHz, DMSO-d₆): 167.6, 164.5, 140.2, 137.4, 137.3, 128.3,128.1, 125.2, 36.6, 34.7, 32.7, 32.6, 31.2, 30.9, 28.8, 28.6, 22.0,13.9.

Example 15 K145 (30)

¹H NMR (400 MHz, DMSO-d₆): 8.08 (brs, 3H), 7.15-7.13 (d, J=8.5 Hz, 2H),7.01 (t, J=7.4 Hz, 1H), 6.86-6.84 (d, J=8.5 Hz, 2H), 3.92 (t, J=6.4 Hz,2H), 3.83 (t, J=6.0 Hz, 2H), 3.00 (m, 2H), 2.76 (t, J=7.3 Hz, 2H),2.53-2.48 (m, 2H), 1.69-1.64 (m, 2H), 1.45-1.39 (m, 2H), 0.92 (t, J=7.3Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆): 167.5, 164.4, 157.1, 137.3, 131.9,129.2, 125.1, 114.3, 66.9, 36.5, 32.9, 32.1, 30.7, 18.6, 13.6.

Example 16 KL-SI-25 (36)

¹H NMR (400 MHz, DMSO-d₆): 8.10 (s, 3H), 7.04 (t, J=7.7 Hz, 1H), 3.85(t, J=6.0 Hz, 2H), 3.04-3.01 (m, 2H), 2.25-2.19 (q, J=7.4 Hz, 2H),1.52-1.47 (m, 2H), 1.27 (m, 10H), 0.86 (t, J=6.7 Hz, 3H), ¹³C NMR (100MHz, DMSO-d₆): 167.6, 164.6, 138.3, 124.9, 36.6, 31.2, 31.1, 28.7, 28.6,28.5, 27.2, 22.0, 13.9.

Example 17 B26

¹H NMR (400 MHz, DMSO-d₆): 8.15 (s, 3H), 7.05 (t, J=7.7 Hz, 1H),3.04-3.00 (m, 2H), 2.20-2.14 (q, J=7.4 Hz, 2H), 1.94 (brs, 3H),1.69-1.59 (m, 6H), 1.47-1.46 (d, J=2.3 Hz, 6H), 1.27-1.23 (m, 2H), ¹³CNMR (100 MHz, DMSO-d₆): 167.5, 164.5, 139.1, 124.3, 41.4, 36.5, 36.4,31.8, 27.9, 25.0.

Example 18 KLII158 (40)

¹H NMR (400 MHz, DMSO-d₆): 7.74 (s, 3H), 7.09-7.07 (d, J=8.5 Hz, 2H),6.84-6.81 (d, J=8.5 Hz, 2H), 4.56-4.53 (m, 1H), 3.91 (t, J=6.5 Hz, 2H),3.72 (t, J=6.2 Hz, 2H), 2.96-2.87 (m, 2H), 2.55 (t, J=7.4 Hz, 2H),2.10-2.02 (m, 1H), 1.88-1.65 (m, 4H), 1.59-1.52 (m, 1H), 1.43-1.35 (m,2H), 1.33-1.28 (m, 6H), 0.87 (t, J=6.8 Hz, 3H); ¹³C NMR (100 MHz,DMSO-d₆): 174.7, 171.5, 156.9, 133.0, 129.2, 114.3, 67.3, 49.6, 36.7,33.6, 31.2, 31.1, 28.7, 28.5, 28.4, 25.5, 22.0, 13.9.

Example 19 KLII159 (39)

¹H NMR (400 MHz, DMSO-d₆): 7.91 (s, 3H), 7.10-7.07 (d, J=8.5 Hz, 2H),6.84-6.82 (d, J=8.5 Hz, 2H), 4.56-4.53 (m, 1H), 3.92 (t, J=6.5 Hz, 2H),3.73 (t, J=6.2 Hz, 2H), 2.99-2.88 (m, 2H), 2.55 (t, J=7.4 Hz, 2H),2.11-2.02 (m, 1H), 1.88-1.63 (m, 4H), 1.61-1.52 (m, 1H), 1.47-1.38 (m,2H), 0.92 (t, J=6.8 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆): 174.7, 171.5,156.9, 133.0, 129.2, 114.3, 67.0, 49.6, 36.6, 33.6, 31.1, 30.8, 28.5,18.7, 13.7.

Example 20 Compound 41

¹H NMR (400 MHz, CDCl3): 11.4 (s, 1H), 8.44 (t, J=5.5 Hz, 1H), 7.08-7.04(m, 3H), 6.84-6.82 (d, J=8.6 Hz, 2H), 3.94-3.89 (m, 4H), 3.69-3.65 (q,J=5.8 Hz, 2H), 2.78 (t, J=7.4 Hz, 2H), 2.52-2.47 (q, J=7.6 Hz, 2H)1.79-1.75 (m, 2H), 1.48-1.34 (m, 20H), 1.32-1.30 (m, 6H), 0.88 (t, J=6.8Hz, 3H).

Example 21 1-(2-aminoethyl)-3-(3-cyclohexylpropylidene)indolin-2-one(S-II-36)

¹H NMR (400 MHz, DMSO): δ 8.1279 (s, 3H), 7.65-7.63 (d, J=7.48, 1H),7.35-7.31 (t, J=7.2 Hz, 1H), 7.22-7.20 (d, J=7.72 Hz, 1H), 7.11-7.07 (t,J=8.16 Hz, 1H), 6.90-6.86 (t, J=7.76 Hz, 1H), 4.01-3.98 (t, J=6.48, 2H),3.03-3.00 (m, 2H), 2.71-2.65 (q, J=7.64 Hz, 2H), 1.74-1.51 (m, 5H),1.49-1.39 (q, J=7.56 Hz, 2H), 1.26-1.11 (m, 4H), 0.97-0.88 (m, 2H)

¹³C NMR (100 MHz, DMSO): 167.1, 142.2, 142.2, 128.9, 128.4, 126.8,123.4, 122.2, 121.8, 108.7, 36.8, 36.7, 35.5, 32.6, 30.7, 26.1, 25.7

Example 22 1-(2-aminoethyl)-3-(3-cyclopropylpropylidene)indolin-2-one(S-II-40)

¹H NMR (400 MHz, MeOD): δ 7.35-7.33 (d, J=6.88 Hz, 1H), 7.35-7.33 (t,J=6.84 Hz, 1H), 7.16-7.07 (m, 3H), 4.11-4.08 (t, J=6.00 Hz, 2H),3.27-3.24 (t, J=5.72 Hz, 3H) 2.88-2.83 (q, J=7.44 Hz, 2H), 1.60-1.55 (q,J=7.16 Hz, 2H), 0.51-0.46 (m, 2H), 0.16-0.12 (m, 2H)

¹³C NMR (100 MHz, DMSO): 170.5, 144.4, 143.1, 130.2, 128.5, 125.1,124.0, 123.7, 109.4, 39.3, 38.7, 34.8, 30.6, 11.7, 5.2.

Example 233-(3-((3r,5r,7r)-adamantan-1-yl)propylidene)-1-(2-aminoethyl)indolin-2-one(S-II-73)

¹H NMR (400 MHz, DMSO): δ 8.11 (s, 1H), 7.62-7.61 (d, J=7.52 Hz, 1H),7.35-7.31 (t, J=7.72 Hz, 1H), 7.22-7.20 (d, J=7.84 Hz, 1H), 7.12-7.09(t, J=7.52 Hz, 1H), 6.91-6.87 (t J=7.84 Hz, 1H), 4.00-3.97 (t, J=6.56,2H), 3.04-3.00 (q, J=5.44, 2H), 2.65-2.59 (q, J=8.04 Hz, 2H), 1.99 (s,3H), 1.71-1.60 (q, J=12.04 Hz, 6H), 1.54 (s, 6H), 1.36-1.32 (m, 2H).

¹³C NMR (100 MHz, DMSO): 167.0, 157.1, 142.1, 140.9, 132.4, 129.3,129.0, 127.2, 123.5, 122.2, 121.6, 114.4, 114.3, 108.7, 67.0, 66.3,37.0, 36.6, 32.8, 30.8, 30.4, 18.7, 13.7.

Example 24 1-(2-aminoethyl)-3-nonylideneindolin-2-one (S-II-103)

¹H NMR (400 MHz, DMSO): δ7.96 (s, 1H), 7.67-7.65 (d, J=7.56, 1H),7.34-7.17 (t, J=7.68, 1H), 7.17-7.16 (d, J=7.8, 3H), 7.11-7.07 (t,J=7.56, 1H), 6.91-6.87 (t, J=7.80, 1H), 3.99-3.96 (t, J=6.32, 2H),2.71-2.65 (q, J=7.40, 2H), 1.61-1.57 (m, 2H), 1.39-1.26 (m, 11H),0.87-0.83 (t, J=6.60, 3H).

¹³C NMR (100 MHz, DMSO): 142.1, 129.0, 127.0, 123.5, 122.2, 121.7, 36.8,31.2, 28.8, 28.6, 28.0, 22.0, 13.9.

Example 251-(2-aminoethyl)-3-(3-(4-methoxyphenyl)propylidene)indolin-2-one(S-II-71)

¹H NMR (400 MHz, DMSO): δ 8.14 (s, 1H), 7.65-7.64 (d, J=7.48, 1H),7.35-7.31 (t, J=7.72, 1H), 7.24-7.20 (t, J=8.48, 3H), 7.10-7.06 (t,J=7.60, 1H), 6.89-6.85 (m, 3H), 4.00-3.96 (t, J=6.52, 2H), 3.01-2.95 (m,4H), 2.88-2.85 (t, J=6.92, 2H), 2.51-2.49 (m, 3H) ¹³C NMR (100 MHz,DMSO): 167.0, 157.7, 142.1, 141.9, 140.9, 140.6, 132.6, 129.3, 129.2,129.1, 128.7, 127.2, 123.5, 122.2, 121.8, 121.6, 119.4, 113.8, 108.7,108.4, 55.0, 37.0, 36.8, 36.8, 36.6, 33.4, 32.8, 30.4, 30.4, 29.3

Example 261-(2-aminoethyl)-3-(3-(4-ethoxyphenyl)propylidene)indolin-2-one(S-II-102)

¹H NMR (400 MHz, DMSO): δ 8.14 (s, 1H), 7.65-7.64 (d, J=7.48, 1H),7.33-7.31 (t, J=7.72 Hz, 1H), 7.24-7.20 (m, 3H), 7.10-7.06 (t, J=7.60Hz, 1H), 6.89-6.85 (m, 3H), 4.00-3.96 (t, J=6.52, 2H), 4.00-3.96 (t,J=6.52 Hz, 3H), 3.01-2.95 (m, 4H), 2.88-2.85 (t, J=6.92 Hz, 2H),2.51-2.49 (m, 3H)

³C NMR (100 MHz, DMSO): 167.1, 142.1, 132.4, 129.3, 129.0, 127.2, 123.6,121.7, 114.2, 108.6, 62.9, 37.1, 36.8, 32.8, 30.4, 14.7

Example 271-(2-aminoethyl)-3-(3-(4-butoxyphenyl)propylidene)indolin-2-one(S-II-78)

¹H NMR (400 MHz, DMSO): δ 8.16 (s, 1H), 7.66-7.64 (d, J=7.48 Hz, 1H),7.35-7.31 (t, J=7.72 Hz, 1H), 7.22-7.16 (m, 3H), 7.08-7.07 (t, J=6.92Hz, 1H), 6.89-6.84 (m, 3H), 4.00-3.97 (t, J=6.56 Hz, 2H), 3.94-3.91 (t,J=6.44 Hz, 3H), 3.03-2.95 (m, 4H), 2.87-2.84 (t, J=6.96, 2H), 1.70-1.63(m, 2H), 1.45-1.39 (m, 2H), 0.94-0.90 (t, J=7.36 Hz, 3H)

¹³C NMR (100 MHz, DMSO): 167.1, 142.9, 142.1, 128.9, 126.5, 123.3,122.2, 121.7, 108.7, 66.3, 108.7, 37.0, 36.7, 36.6, 36.5, 32.0, 28.0,22.4

Example 281-(2-aminoethyl)-3-(3-(4-(heptyloxy)phenyl)propylidene)indolin-2-one(S-II-103)

¹H NMR (400 MHz, DMSO): δ 8.06 (s, 1H), 7.66-7.64 (d, J=7.48 Hz, 1H),7.35-7.31 (t, J=7.72 Hz, 1H), 7.22-7.18 (m, 3H), 7.10-7.06 (t, J=7.56Hz, 1H), 6.89-6.84 (m, 3H), 3.99-3.95 (t, J=6.32, 2H), 3.93-3.90 (t,J=6.52 Hz, 3H), 3.02-2.95 (m, 4H), 2.87-2.84 (t, J=7.04 Hz, 2H),1.72-1.65 (m, 2H), 1.43-1.17 (m, 8H), 0.88-0.85 (t, J=6.64 Hz, 3H)

³C NMR (100 MHz, DMSO): 167.0, 157.1, 142.1, 140.9, 132.4, 129.3, 129.0,127.2, 123.6, 122.2, 121.7, 114.4, 108.6, 37.0, 36.7, 32.8, 31.2, 30.4,28.7, 28.7, 28.4, 25.5, 22.0, 13.9.

Example 291-(2-aminoethyl)-3-(3-(4-fluorophenyl)propylidene)indolin-2-one(S-II-39)

¹H NMR (400 MHz, MeOD): δ 7.66-7.64 (d, J=7.56, 1H), 7.35-7.27 (m, 3H),7.13-6.97 (m, 5H), 7.10-7.06 (t, J=6.96 Hz, 1H), 6.90-6.86 (t, J=7.24Hz, 1H), 4.09-4.06 (t, J=5.88 Hz, 2H), 3.26-3.23 (t, J=6.12 Hz, 2H),3.08-3.03 (m, 2H), 2.99-2.95 (m, 2H).

¹³C NMR (100 MHz, DMSO): 170.0, 164.2, 161.8, 143.1, 138.0, 131.2,130.4, 129.0, 125.0, 124.1, 123.5, 116.3, 116.1, 109.5, 39.3, 38.7,34.6, 32.1

Example 301-(2-aminoethyl)-3-(3-(4-chlorophenyl)propylidene)indolin-2-one(S-II-60)

¹H NMR (400 MHz, DMSO): δ 7.97 (s, 3H), 7.67-7.65 (d, J=7.52 Hz, 1H),7.36-7.32 (m, 5H), 7.17-7.15 (d, J=7.80 Hz, 2H), 7.10-7.06 (t, J=6.96Hz, 1H), 6.87-6.84 (t, J=7.04 Hz, 1H), 3.98-3.94 (t, J=6.28 Hz, 2H),3.04-2.99 (m, 4H), 2.95-2.91 (m, 2H).

¹³C NMR (100 MHz, DMSO): 140.4, 139.8, 130.7, 129.1, 128.3, 127.4,123.6, 122.2, 121.6, 108.7, 37.1, 36.7, 33.0, 29.9

Example 311-(2-aminoethyl)-3-(3-(4-nitrophenyl)propylidene)indolin-2-one (S-II-61)

¹H NMR (400 MHz, MeOD): δ 8.75-8.74 (d, J=7.48 Hz, 3H), 7.67-7.65 (d,J=7.52 Hz, 1H), 7.35-7.2890 (m, 5H), 7.23-7.20 (m, 2H), 7.10-7.06 (t,J=6.96 Hz, 1H), 6.90-6.86 (t, J=7.24 Hz, 1H), 4.00-3.96 (t, J=6.48 Hz,2H), 3.05-2.99 (m, 4H), 2.95-2.91 (m, 2H).

¹³C NMR (100 MHz, DMSO): δ 168.7, 147.4, 145.1, 135.1, 131.2, 129.9,123.8, 124.2, 123.2, 115.6, 109.5, 37.9, 38.7, 26.6, 32.1

Example 32 In Vitro Test SphK1 and SphK2 Test

The results from FIGS. 1-6 demonstrate that Formula III (K145) and someother compounds of Formula I and Formula II significantly inhibit SphK2,but not SphK1, thus demonstrating they are selective SphK2 inhibitors.As shown in FIG. 1, Formula III (K145) dose-dependently inhibited SphK2with a IC₅₀ of 5 uM. Significantly, the results of FIG. 2 demonstratesthat the potency of this type of SphK2 inhibitors from Formula I can besignificantly improved by structural modification as Formula IV(KLIII16) with a longer alkyl chain has an IC₅₀ of 1.9 uM compared tothat of Formula III (K145).

Kinetic Studies of Formula III (K145)

As shown in FIG. 7, the kinetic studies of Formula III revealed thatFormula III (K145) is a competitive inhibitor (with the substratesphingosine) of SphK2 with a Ki value of 4.3±0.7M.

Cell Viability Assays in Human Leukemia U937 Cells

Cells were cultured at a density of 5×10⁴ (U937) or 1×10⁴ (PC-3, DU145,M12, HT29) cells per well in flat bottomed 96-well plates and treatedwith various concentrations of test compound at 37° C. (5% CO₂). After24 h, 20 μL, of CellTiter 96® Aqueous One Solution Reagent (Promega,Madison, Wis.) was added to each well according to the manufacturer'sinstructions. After 1 hour, the cell viability was determined bymeasuring the absorbance at 490 nm using a micro-plate reader.

The results are presented in FIG. 8. These results show that Formula III(K145) inhibited the proliferation of tested cancer cells with an IC₅₀at single digit micromolar concentrations.

The results of Table 1 also show that Formula II compounds inhibited theproliferation of human leukemia U937 cells.

TABLE 1 Inhibition of U937 cell proliferation by indicated compounds.*Compound IC₅₀ ± SEM Compound IC₅₀ ± SEM Compound IC₅₀ ± SEM S-II-1292.128 ± 0.006 S-II-103 3.5 ± 0.3 S-II-104 3.9 ± 0.3 S-II-36 2.7 ± 0.2S-II-71 0.92 ± 0.04 S-II-39 2.69 ± 0.06 S-II-40 1.16 ± 0.05 S-II-1020.95 ± 0.11 S-II-60 4.0 ± 0.7 S-II-73 4 ± 1 S-II-78 4.6 ± 0.8 S-II-613.6 ± 0.2 *U937 cells were treated with indicated compounds at variousconcentrations for 24 hrs, after which cell viability was measured usingMTT assay and IC₅₀ was calculated.

Western Blot Analysis

Cells (5×10⁵ per ml) were treated with various concentrations of testcompound at 37° C. (5% CO₂) for 3 hrs, then stimulated with TPA at afinal concentration of 200 nM for 20 min. Samples from whole-cellpellets were prepared and 30 μg protein for each condition was subjectedto SDS-PAGE, transferred onto a PVDF membrane, and blocked with 5%fat-free milk for 30 min. The membrane is probed with primary antibodiesovernight at 4° C. followed by incubation with horseradishperoxidase-labeled anti-mouse IgG (1:5000, BD Bioscience). Theimmunoreactive bands are detected by chemilluminescence methods (Pierce)and visualized on Kodak Omat film. The following primary antibodies wereused: phospho-p44/42 MAPK (ERK1/2, Thr202/Tyr204), p44/42 MAPK,phospho-p90RSK (Thr359/Ser363), RSK1/RSK2/RSK3 (Cell Signaling). Blotswere reprobed with antibodies against α-tubulin to ensure equal loadingand transfer of proteins.

The results are presented in FIG. 9, which shows that Formula III (K145)significantly inhibited the phosphorylation of both ERK and Akt at 3 μMconcentrations. The results are consistent with reported results thatSphK execute their effects through, at least through, the Raf/MEK/ERKand PI3K/Akt signaling pathways. As shown in FIG. 10, compounds S-II-71and S-II-103 also inhibit the Raf/MEK/ERK and PI3K/Akt signalingpathways, which indicates that inhibition of SphK2 by compounds fromFormula II, interferes with the Raf/MEK/ERK and PI3K/Akt signalingpathways.

In Vitro Kinase Screening Cell Apoptosis Assays.

Apoptosis was measured by flow cytometry using annexin V/propidiumiodide (PI) as staining reagent. Briefly, after treatment with testcompound of varying concentrations for varying intervals (4, 8, 18, 36hrs), cells were washed twice with cold PBS and then resuspended in 1×binding buffer (10 mM HEPES[N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid]/NaOH, pH 7.4, 140mM NaOH, 2.5 mM CaCl₂). The cells were then incubated with annexinV—fluorescein isothiocyanate (FITC) (BD PharMingen, San Diego, Calif.)and 5 μg/mL propidium iodide (PI), and incubated for 15 minutes at roomtemperature in the dark per the manufacturer's instructions. The sampleswere analyzed by flow cytometry using a Becton Dickinson FACScan (BectonDickinson, San Jose, Calif.) within 1 hr to determine the percentage ofcells displaying annexin V staining (early apoptosis) or both annexin Vand PI staining (late apoptosis).

The results are presented in FIG. 11, which shows that Formula III(K145) significantly and dose-dependently induced apoptosis in U937cells. After 18 hrs, Formula III (K145) significantly induced apoptosisin U937 cells at 5 μM. At 10 μM concentration, Formula III (K145)significantly induced apoptosis at as early as 4 hrs treatment. As shownin FIG. 12, S-II-71 and S-II-103 from Formula II also induced apoptoticeffects in U937 cells after 24 hrs treatment. Interestingly, S-II-71induced both early and late apoptotic effect while S-II-103 mainlyinduced late apoptotic effects.

Example 18 In Vivo Studies Anti-Proliferation of U937 Xenografts in NudeMice.

Female nude mice (BALB/c-nu, n=7) were inoculated with 3×10⁷ U937 cellssubcutaneously in the right flank. After 7 days of implant of U937cells, mice were treated daily with Vehicle (control), or Tamibarotene(15 mg/Kg, positive control), or Formula III (K145) (15 mg/Kg) by i.p.administration. Tumor size was measured every other day. After 18 daystreatment, mice were sacrificed and the tumor were removed and weighed.

The results are presented in FIG. 13A, K145 significantly inhibited thegrowth of U937 tumors in nude mice with a TGI of 44.2%, slightly lesspotent than tamibarotene (TGI=50.4%) after 17 days treatment. This isalso reflected by the tumor weights of treatment groups (FIG. 13B). Thetumor growth curve during the treatment course (FIG. 13C) also atteststo the anti-tumor effects of K145 in this model. Lastly, as shown inFIG. 13D, the body weights of K145-treated mice remained the same asthat of vehicle-treated mice, while tamibarotene treatment caused bodyweight decreases in the mice. These results strongly suggest that K145exhibits comparable in vivo anti-tumor activity to tamibarotene, whileconcomitantly exhibiting less toxicity in this U937 xenograft cancermodel.

Anti-Growth of JC Xenografts in Syngenic BALB/c Mice.

Female BALB/c mice (n=12) were injected with 1×10⁶ JC cellssubcutaneously in the flank, and treatment with the compound produced asdescribed in Example 15 i.e. 3-(2-aminoethyl)-5-[3-(4-butoxyphenyl)-propylidene]-thiazolidine-2,4-dione(Formula III-K145, 20 mg/kg and 35 mg/Kg dosage daily (i.p.) was startedseven days after the tumor cell injection and stopped at day 15 aftertumor injection. Mice were sacrificed at 15 days treatment.

As illustrated in FIG. 14A, treatment of BALB/c mice (n=8) bearing theJC xenograft significantly inhibited tumor growth at both doses with thehigher dose being more potent. After 15 days treatment, the mean volumeof the JC tumors in the treated-mice at both doses was >50% smaller thanthat in the vehicle-treated mice. Tumor weights of K145-treated micewere also significantly less than that in vehicle-treated mice in adose-dependent manner (FIG. 14B). Post-experiment visual evaluation ofthe tumor samples also confirms the results (FIG. 14D). We analyzed thetumor samples to detect K145, the change of S1P by ESI-MS/MS and thechange of signaling pathways by Western blot. As shown in FIG. 14C, K145was detected in JC tumors and the S1P level was suppressed compared tovehicle, consistent with the results from U937 cells assays. Notably,the p-ERK and p-Akt levels were decreased in the tumor samples comparedto the vehicle controls (FIG. 14E), which is consistent with the resultsfrom U937 cells (FIG. 9). We did not observe significant changes in bodyweights and the major organs, such as heart, lung, liver and kidney,thus indicating a lack of general toxicity of K145.

Anti-Proliferation of U937 Xenografts in Nude Mice by K145 Through OralAdministration.

We examined the anticancer activity of K145 to inhibit the tumor growthof U937 cells in nude mice (BALB/c-nu) by oral administration toinvestigate whether it is orally available. In this experiment, K145 wasgiven at 50 mg/kg by oral gavage daily for 15 days and tumor volume andanimal weights were measured every other day. Again, tamibarotene (20mg/kg) was used as positive control. As shown in FIG. 15A, tumor weightsof K145-treated mice were significantly less than that invehicle-treated mice and K145 exhibited better antitumor activity thantamibarotene at tested doses by oral administration (TGI for K145 andtamibarotene are 51.25% and 33.37%, respectively). Visual examination ofthe tumor samples also confirmed the significant inhibition of U937tumor growth by K145 (FIG. 15B). Tumor growth curve also demonstratedthe superior anti-tumour activity of K145 in these experimental settings(FIG. 15C). As shown in FIG. 15D, at the beginning of the treatment,there was a slight decrease of body weights in K145-treated group butthe body weights of this group came back in the remaining course of thestudy. Collectively, the results of in vivo studies with K145 by oraladministration demonstrated that K145 is orally available to inhibit thegrowth of U937 tumors at 50 mg/kg dose and no apparent toxicity wasobserved, which is consistent with the results from in vivo studies byi.p. injection administration.

Example 19 In Vitro Target Validation of K145

Next we examined whether K145 affects cellular levels of S1P. Humanleukemia U937 cells have been demonstrated to be a good model to testcompounds that interfere with the SphK/S1P system and it has previouslybeen shown that S1P is protective against apoptosis of U937 cells.Therefore, we further characterized K145 in U937 cells. As shown in FIG.16A, K145 is readily taken up by U937 cells in a concentration dependentmanner. As shown in FIGS. 16B and 16C, treatment with K145 (10 μM)caused a decrease of total cellular S1P without significant effects onceramide levels. The inhibitory potency of K145 on cellular level of S1Pis somewhat less than its IC₅₀ (4.3 μM) determined at recombinant SphK2.This might be due to the fact that many enzymes such as SphK1, SphK2,S1P lyase, and S1P phosphatise, not just SphK2, are involved in theregulation of cellular S1P. The level of ceramide-1-phosphate (C1P) wasnot significantly affected upon treatment with K145 (10 μM, FIG. 16D),which indicates that K145 does not interfere with CERK and/or ceramidesynthase, consistent with the results from recombinant CERK studies. Tofurther confirm its SphK2 selectivity, we then tested the effects ofK145 on the phosphorylation of FTY720, a SphK2 specific substrate [35].As shown in FIG. 16E, K145 inhibited the phosphorylation of FTY720,which further indicates the SphK2 specificity of K145.

We claim:
 1. A compound of Formula I:

wherein, R₁ is selected from the group consisting of: C₃-C₁₄ alkyl andC₃-C₁₄ alkoxyl; R₂, R₃, R₄ and R₅ may be the same or different and areindependently selected from: H, C₁-C₈ alkyl, C₁-C₈ alkoxyl, C₁-C₈alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; V is S, O,NH, or CH₂; X is C₁-C₄ alkyl; Y is C₁-C₄ alkyl; Z is S or O or NR⁶ inwhich R⁶ is selected from the group consisting of: H, C₁-C₈ alkyl, orisopropyl, tert-butyl, a saturated or unsaturated monocyclic ring withring size ranging from 3-7 carbons per ring, and phenyl which may besubstituted with one or more substituents selected from the groupconsisting of: C₁-C₈ alkyl, C₁-C₈ alkoxyl, C₁-C₈ alkylcarbonyl, halogen,hydroxyl, amino, nitro, and cyano; and W is NR₇R₈ where R₇ and R₈ may bethe same or different and are independently selected from H; C₁-C₄alkyl; a saturated heterocycle comprising N bonded directly to Y; and anunsubstituted or substituted guanidine moiety.
 2. The compound of claim1, wherein the number of carbon atoms in said saturated or unsaturatedmonocyclic ring with ring size from 3-7 is selected from the groupconsisting of 3, 4, 5, 6, and
 7. 3. The compound of claim 1, whereinsaid saturated heterocycle is selected from the group consisting ofmorpholine, piperidine, piperazine, and pyrrolidine.
 4. The compound ofclaim 1, wherein said compound is selected from3-(2-aminoethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dioneand3-(2-aminoethyl)-5-[3-(4-octyl-phenyl)-propylidene]-thiazolidine-2,4-dione.5. A compound of Formula II:

wherein, R₁₀ is selected from the group consisting of: C₃-C₁₄ alkyl,C₃-C₁₄ alkoxyl; R₁₁, R₁₂, R₁₃ and R₁₄ may be the same or different andare independently selected from: H, C₁-C₈ alkyl, C₁-C₈ alkoxyl, C₁-C₈alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; R₁₅, R₁₆, R₁₇and R₁₈ may be the same or different and are independently selected fromthe group consisting of: C₁-C₈ alkyl, C₁-C₈ alkoxyl, C₁-C₈alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; X is C₁-C₄alkyl; Y is C₁-C₄ alkyl; and W is NR¹⁹R²⁰ where R¹⁹ and R²⁰ may be thesame or different and are selected from the group consisting of: H,C₁-C₄ alkyl; a saturated heterocycle comprising N bonded directly to Y,and an unsubstituted or substituted guanidine moiety.
 6. The compound ofclaim 5, wherein the number of carbon atoms in said saturated orunsaturated monocyclic ring with ring size from 3-7 is selected from thegroup consisting of 3, 4, 5, 6, and
 7. 7. The compound of claim 5,wherein said saturated heterocycle is selected from the group consistingof morpholine, piperidine, piperazine, and pyrrolidine.
 8. A method oftreating diseases or conditions associated with positive SphK2 activityin a patient in need thereof, comprising the step of administering tosaid patient a sufficient quantity of at least one compound of Formula Ior Formula II:

wherein, in Formula I: R₁ is selected from the group consisting of:C₃-C₁₄ alkyl and C₃-C₁₄ alkoxyl; R₂, R₃, R₄ and R₅ may be the same ordifferent and are independently selected from: H, C₁-C₈ alkyl, C₁-C₈alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino, nitro, andcyano; V is S, O, NH, or CH₂; X is C₁-C₄ alkyl; Y is C₁-C₄ alkyl; Z is Sor O or NR⁶ in which R⁶ is selected from the group consisting of: H,C₁-C₈ alkyl, or isopropyl, tert-butyl, a saturated or unsaturatedmonocyclic ring with ring size ranging from 3-7 carbons per ring, andphenyl which may be substituted with one or more substituents selectedfrom the group consisting of: C₁-C₈ alkyl, C₁-C₈ alkoxyl, C₁-C₈alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; and W isNR₇R₈ where R₇ and R₈ may be the same or different and are independentlyselected from H; C₁-C₄ alkyl; a saturated heterocycle comprising Nbonded directly to Y; and an unsubstituted or substituted guanidinemoiety; and wherein in Formula II: R₁₀ is selected from the groupconsisting of: C₃-C₁₄ alkyl, C₃-C₁₄ alkoxyl; R₁₁, R₁₂, R₁₃ and R₁₄ maybe the same or different and are independently selected from: H, C₁-C₈alkyl, C₁-C₈ alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino,nitro, and cyano; R₁₅, R₁₆, R₁₇ and R₁₈ may be the same or different andare independently selected from the group consisting of: C₁-C₈ alkyl,C₁-C₈ alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino, nitro, andcyano; X is C₁-C₄ alkyl; Y is C₁-C₄ alkyl; and W is NR¹⁹R²⁰ where R¹⁹and R²⁰ may be the same or different and are selected from the groupconsisting of: H, C₁-C₄ alkyl; a saturated heterocycle comprising Nbonded directly to Y, and an unsubstituted or substituted guanidinemoiety.
 9. The method of claim 8, wherein the number of carbon atoms insaid saturated or unsaturated monocyclic ring with ring size from 3-7 isselected from the group consisting of 3, 4, 5, 6, and
 7. 10. The methodof claim 8, wherein said saturated heterocycle is selected from thegroup consisting of morpholine, piperidine, piperazine, and pyrrolidine.11. The compound of claim 8, wherein said compound is selected from3-(2-aminoethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dioneand3-(2-aminoethyl)-5-[3-(4-octyl-phenyl)-propylidene]-thiazolidine-2,4-dione.12. The method of claim 8, wherein said disease or condition associatedwith positive SphK2 activity is selected from the group consisting of:cancer, arthrosclerosis, arthritis, diabetes, obesity, osteoporosis,inflammatory diseases and Alzheimer's disease.
 13. A method ofinhibiting SphK2, comprising the step of exposing said SphK2 to at leastone compound of Formula I or Formula II:

wherein, in Formula I: R₁ is selected from the group consisting of:C₃-C₁₄ alkyl and C₃-C₁₄ alkoxyl; R₂, R₃, R₄ and R₅ may be the same ordifferent and are independently selected from: H, C₁-C₈ alkyl, C₁-C₈alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino, nitro, andcyano; V is S, O, NH, or CH₂; X is C₁-C₄ alkyl; Y is C₁-C₄ alkyl; Z is Sor O or NR⁶ in which R⁶ is selected from the group consisting of: H,C₁-C₈ alkyl, or isopropyl, tert-butyl, a saturated or unsaturatedmonocyclic ring with ring size ranging from 3-7 carbons per ring, andphenyl which may be substituted with one or more substituents selectedfrom the group consisting of: C₁-C₈ alkyl, C₁-C₈ alkoxyl, C₁-C₈alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; and W isNR₇R₈ where R₇ and R₈ may be the same or different and are independentlyselected from H; C₁-C₄ alkyl; a saturated heterocycle comprising Nbonded directly to Y; and an unsubstituted or substituted guanidinemoiety; and wherein in Formula II: R₁₀ is selected from the groupconsisting of: C₃-C₁₄ alkyl, C₃-C₁₄ alkoxyl; R₁₁, R₁₂, R₁₃ and R₁₄ maybe the same or different and are independently selected from: H, C₁-C₈alkyl, C₁-C₈ alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino,nitro, and cyano; R₁₅, R₁₆, R₁₇ and R₁₈ may be the same or different andare independently selected from the group consisting of: C₁-C₈ alkyl,C₁-C₈ alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino, nitro, andcyano; X is C₁-C₄ alkyl; Y is C₁-C₄ alkyl; and W is NR¹⁹R²⁰ where R¹⁹and R²⁰ may be the same or different and are selected from the groupconsisting of: H, C₁-C₄ alkyl; a saturated heterocycle comprising Nbonded directly to Y, and an unsubstituted or substituted guanidinemoiety.
 14. The method of claim 13, wherein the number of carbon atomsin said saturated or unsaturated monocyclic ring with ring size from 3-7is selected from the group consisting of 3, 4, 5, 6, and
 7. 15. Themethod of claim 13, wherein said saturated heterocycle is selected fromthe group consisting of morpholine, piperidine, piperazine, andpyrrolidine.
 16. The compound of claim 13, wherein said compound isselected from3-(2-aminoethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dioneand3-(2-aminoethyl)-5-[3-(4-octyl-phenyl)-propylidene]-thiazolidine-2,4-dione.17. The method of claim 13, wherein said SphK2 is present in a cell. 18.The method of claim 17, wherein said cell is selected but not limited toa cancer cell, cardiocyte cell, epithelial cell, pancreatic cell, andneuronal cell, and said method includes a step of exposing said cell tosaid at least one compound of Formula I or Formula II.
 19. A method ofinhibiting growth or killing or damaging cancer cells, comprising thestep of exposing said cancer cells to a compound of Formula I or FormulaII:

wherein, in Formula I: R₁ is selected from the group consisting of:C₃-C₁₄ alkyl and C₃-C₁₄ alkoxyl; R₂, R₃, R₄ and R₅ may be the same ordifferent and are independently selected from: H, C₁-C₈ alkyl, C₁-C₈alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino, nitro, andcyano; V is S, O, NH, or CH₂; X is C₁-C₄ alkyl; Y is C₁-C₄ alkyl; Z is Sor O or NR⁶ in which R⁶ is selected from the group consisting of: H,C₁-C₈ alkyl, or isopropyl, tert-butyl, a saturated or unsaturatedmonocyclic ring with ring size ranging from 3-7 carbons per ring, andphenyl which may be substituted with one or more substituents selectedfrom the group consisting of: C₁-C₈ alkyl, C₁-C₈ alkoxyl, C₁-C₈alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; and W isNR₇R₈ where R₇ and R₈ may be the same or different and are independentlyselected from H; C₁-C₄ alkyl; a saturated heterocycle comprising Nbonded directly to Y; and an unsubstituted or substituted guanidinemoiety; and wherein in Formula II: R₁₀ is selected from the groupconsisting of: C₃-C₁₄ alkyl, C₃-C₁₄ alkoxyl; R₁₁, R₁₂, R₁₃ and R₁₄ maybe the same or different and are independently selected from: H, C₁-C₈alkyl, C₁-C₈ alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino,nitro, and cyano; R₁₅, R₁₆, R₁₇ and R₁₈ may be the same or different andare independently selected from the group consisting of: C₁-C₈ alkyl,C₁-C₈ alkoxyl, C₁-C₈ alkylcarbonyl, halogen, hydroxyl, amino, nitro, andcyano; X is C₁-C₄ alkyl; Y is C₁-C₄ alkyl; and W is NR¹⁹R²⁰ where R¹⁹and R²⁰ may be the same or different and are selected from the groupconsisting of: H, C₁-C₄ alkyl; a saturated heterocycle comprising Nbonded directly to Y, and an unsubstituted or substituted guanidinemoiety.
 20. The method of claim 19, wherein the number of carbon atomsin said saturated or unsaturated monocyclic ring with ring size from 3-7is selected from the group consisting of 3, 4, 5, 6, and
 7. 21. Themethod of claim 19, wherein said saturated heterocycle is selected fromthe group consisting of morpholine, piperidine, piperazine, andpyrrolidine.
 22. The compound of claim 19, wherein said compound isselected from3-(2-aminoethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dioneand3-(2-aminoethyl)-5-[3-(4-octyl-phenyl)-propylidene]-thiazolidine-2,4-dione.23. The method of claim 19, wherein said cancer cells are of a typeselected from the group consisting of: leukemia, lymphoma, sarcoma,neuroblastoma, lung cancer, skin cancer, head squamous cell carcinoma,neck squamous cell carcinoma, prostate cancer, colon cancer, breastcancer, ovarian cancer, cervical cancer, brain cancer, bladder cancer,and pancreatic cancer.